Diagnostic agent and method for the detection of occult blood in feces

ABSTRACT

A diagnostic agent for the detection of occult blood in feces comprising hydrogen peroxide and, as a chromogen, guaiaconic acid A, optionally with a stabilizer. The invention provides, as a novel substance, guaiaconic acid A having a specific extinction E 1  cm 1%  at 600 nm of at least 200, determined by the reaction with peroxidase and hydrogen peroxide, having infra-red bands at 
     1600 cm -1  (m); 
     1505 cm -1  (v.s.); 
     1260 cm -1  (s;b); 
     1115 cm -1  (m); 
     1025 cm -1  (m); and 
     1200 cm -1  (s;b) 
     and having an R F  value of 0.45 (toluene/dioxan/glacial acetic acid; 90:25:10 v/v/v).

The present invention is concerned with guaiaconic acid A and with thepreparation thereof. In different aspect, the invention provides adiagnostic agent for the detection of occult blood in feces which, asthe chromogen therein, contains guaiaconic acid A with a specificextinction E₁ cm^(1%) at 600 of at least 200.

The detection of occult blood in feces is important for the diagnosis ofdiseases of the digestive system which are accompanied by hemorrhages.Hemorrhages in the gastro-intestinal tract are usually caused by ulcers,polyps and, in particular tumors. Thus, the detection of blood in fecesis an important adjunct for the early diagnosis of carcinomas of thedigestive system.

For a long time, the guaiac test has been used for this diagnosis,catalysis by the peroxidate-active blood components thereby beingutilized. The oxygen liberated from hydrogen peroxide is herebytransferred to a chromogen which is oxidized to a colored material andthus indicates the presence of a peroxidatively-active substance.

The guaiac test is known in several modifications. In general, it iscarried out as follows: to a slurry of feces there is added hydrogenperoxide and an alcoholic solution of guaiac resin, a blue colorationindicating the presence of blood. Recently, a rapid diagnostic agent forthe detection of blood in feces has been described, which comprises afilter paper impregnated with guaiac resin. This filter paper is coatedwith a sample of feces and, for development, an alcoholic solution ofhydrogen peroxide is applied to the rear side thereof. When blood ispresent, a blue ring forms.

However, the use of guaiac resin for the detection of blood in feces isnot without problems. In the case of the test tube test, falselypositive results are frequently found, whereas in the case of the rapidtest, the detection limits depend very largely upon the other componentsof the faecal sample. This is due to the nature of the guaiac resin.

Guaiac resin is obtained from the heartwood of the tropical treesGuajacum officinalic and Guajacum sanctum and comprises a number ofcomponents. In principle, for the coloration by hydrogen peroxide in thepresence of peroxidate-active substances, such as blood and peroxidase,two main components are responsible, namely, furoguaiacin and guaiaconicacid, especially the so-called substance A (cf. H. Auterhoff et al.,Arch. Pharm., 299, 618/1966 and 302, 545/1969). In addition to a fewother compounds which are present in small amounts and which can also beoxidized, there is also a comparatively large number of other componentmaterials which do not influence the indicator reaction but whichpossibly inhibit it. These include, for example, guaiaretic acid,dihydroguaiaretic acid, guaiacic acid, vanillin and others.

It is, of course, well known that, in a natural product, all thecomponents do not always occur in the same ratio so that naturallyoccurring guaiac resin also has a variable composition, depending uponthe isolation procedure used. Furthermore, it will also be readilyappreciated that, in the course of impregnation with guaiac resin, theunstable components of the mixture can also be decomposed and thus,again, can form a mixture of components of varying composition.

In the case of rapid diagnostic agents, which have recently achievedgreat importance, one of the main prerequisities for their practical useis that they can always be produced exactly and reproducably. However,this prerequisite was not satisfied in the case of the previouslydescribed diagnostic agents.

Thus, the problem exists of providing, by the use of definite andsufficiently pure reagents, a rapid diagnostic agent for the detectionof occult blood in feces which can be produced exactly and reproducably,can be analytically tested and is also sufficiently stable.

Surprisingly, we have now found that by using guaiaconic acid A, one ofthe main components of crude guaiac resin, as chromogen, a rapiddiagnostic agent is obtained for the determination of occult blood infeces which possesses the above-mentioned necessary properties and,furthermore, has a definite practical limit of detection.

As already mentioned above, guaiac resin consists essentially of twocolor-forming main components, furoguaiacin and guaiaconic acid, thestructure of furoguaiacin having been unambiguously clarified bysynthesis. Experiments have now shown that furoguaiacin cannot be usedas a chromogen for a rapid diagnostic agent for blood in feces becauseit gives test papers which are much too sensitive and are also unstable.

The isolation of the other main component, guaiaconic acid, was,according to the literature, only possible by preparative thin layerchromatography, the substance being obtained in such small amounts thatit could scarcely be practically considered as a chromogen for rapiddiagnostic agents.

Admittedly the literature describes various attempts to isolatecomparatively large amounts of guaiaconic acid but the results obtainedwere not very satisfactory. Thus, for example, in the thesis by J. Kuhlof the Technical High School in Braunschweig (1964, page 21), it isstated that the column chromatographic separation of guaiaconic resinwith silica gel is not possible.

Surprisingly, we have now found that, in spite of this preconceivedview, it is possible to separate guaiac resin by column chromatographywith silica gel and to obtain guaiaconic acid in almost pure form which,without further purification, can be used for diagnostic agents for thedetermination of blood in feces.

Therefore, according to one aspect of the present invention, there isprovided guaiaconic acid (in order to distinguish from the prior art, itis called guaiaconic acid A) which is characterized by a specificextinction E₁ cm.^(1%) at 600 nm of at least 200, determined by thereaction with peroxidase and hydrogen peroxide, by infrared bands at1600 cm⁻¹ (m); 1505 cm⁻¹ (v.s.); 1260 cm⁻¹ (s;b); 1200 cm⁻¹ (s;b); 1115cm⁻¹ (m) and 1025 cm⁻¹ (m); and an R_(F) value of 0.45(toluene/dioxan/glacial acetic acid 90:45:20 v/v/v) and which isobtained by column chromatographic separation on a neutral silica gelwhich has been pretreated with acid, using an appropriate elution agent,for example n-heptane/ethyl acetate or toluene/acetone, under aprotective gas.

As already stated above, the guaiaconic acid A thus obtained is thinlayer chromatographically not quite uniform; i.e. apart from the maincomponent, which becomes blue colored in light or with peroxidase andhydrogen peroxide, it also contains impurities which, however, becauseof their low concentration, can be neglected for the use thereof as achromogen in a diagnostic agent. The guaiaconic acid A according to thepresent invention is amorphous and, as stated above, can becharacterized by bands in the infra-red spectrum, as well as by thespecific extinction and the R_(F) value in the thin layer chromatogram(cf. Example 1 hereof).

Stating the specific extinction for the characterization has proved tobe desirable since the mentioned impurities, as well as traces ofsolvent possibly present, considerably disturb the normal methods ofanalysis, such as the ultra-violet and NMR spectrum.

By the specific extinction, there is to be understood the extinctionwhich is produced by 1 g. guaiaconic acid A in 100 ml. of solution byperoxidase and hydrogen peroxide, measurement being carried out in a 1cm. long cuvette at 600 nm (E₁ cm.^(1%)).

The detailed description of the isolation of guaiaconic acid A givenhereinafter in Example 1 is only a preferred embodiment of the processaccording to the present invention. A separation of accompanyingmaterials of natural guaiac resin by acetone/toluene precipitation is,of course, also possible in other ways, for example by ethanol/tolueneprecipitation; by dissolving in glacial acetic acid and diluting withwater to 30% acetic acid, taking up the precipitate in acetone/toluene(1:5 v/v) of dissolving in methyl isobutyl ketone, extracting with asodium hydroxide/phosphate buffer of pH 13, concentrating the organicphase and diluting with toluene. Furthermore, solvent mixtures, forexample xylene/acetone and methyl isobutyl ketone/toluene, can also beused as elution agents, whereby the amount ratios can also be varied.Furthermore, the throughput of guaiac resin and elution agent used canalso be varied. As protective gas, it is preferable to use nitrogen orcarbon dioxide.

The present invention also provides an improved diagnostic agent for thedetection of occult blood in feces which is analogous to the knownguaiac test but in which, instead of natural guaiac resin, as chromogenthere is used guaiaconic acid A with a specific extinction E₁ cm.^(1%)at 600 nm of at least 200, determined by the reaction with peroxidaseand hydrogen peroxide.

Experiments have, surprisingly, shown that test papers which have beenproduced analogously to the known guaiac test but which, as chromogen,contained guaiaconic acid A, react with "artificial feces" (blood inwater) just as sensitively as the commercially available test papersbased on unpurified guaiac resin. In natural feces to which 1-3% ofblood had been added, however, the papers according to the presentinvention reacted positively, whereas the commercially available testpapers gave variable results, the limit of sensitivity, depending uponthe feces varying from 1-7% and more of blood.

As already stated above, the guaiaconic acid A according to the presentinvention is characterized by its specific extinction. Since, in theprocess of isolating the guaiaconic acid, the purity of the guaiaconicacid A obtained depends upon the guaiac resin used and upon the processconditions employed, the specific extinction of the guaiaconic acid Aobtained can also vary. We have found that the guaiaconic acid A in thediagnostic agent is especially suitable when its specific extinctionexceeds 200. Crude guaiac resin has a value of about 100 which is due tothe variable amounts of guaiaconic acid A, furoguaiacin and otherunknown oxidizable components.

The guaiaconic acid A can be used in the diagnostic agent in amounts offrom 40 to 250 mg. and preferably of from 50 to 150 mg. per 100 ml. ofimpregnation solution when it has a specific extinction of 250. In thecase of other specific extinctions, the values are amendedcorrespondingly.

In recent years, rapid tests have been used more and more in medicalpractice and in clinical laboratories as a diagnostic adjuvant. As arule, they are absorbent carriers, usually papers, which have beenimpregnated with the reagents necessary for the detection reaction andwhich, after immersion into the liquid to be investigated, show a colorreaction. The detection or the semi-quantitative determination ofpathological body components can be carried out therewith quickly andalso be untrained personnel, such as medical auxiliaries.

Apart from the actual reaction components, test papers of this type canalso contain a number of additional materials, for example, buffers,wetting agents, thickening agents, protective colloids, complex formersand stabilizers, depending upon the indicator used and the purpose ofthe test.

Thus, the present invention also provides a test paper for the detectionof occult blood in feces which, as chromogen, contains guaiaconic acid Awith a specific extinction of E₁ cm.^(1%) at 600 nm of at least 200. Inorder to prevent the papers which have been impregnated with theguaiaconic acid A from becoming blue colored due to light and/or air, itis necessary to add thereto an appropriate stabilizer. We have foundthat previously unknown stabilizers of the arylsemicarbazide group ofcompounds prevent the appearance of this blue coloration. Furthermore,we have found that, within certain limits, the sensitivity can bemodified by the addition of these arylsemicarbazides.

The new stabilizers for oxidation indicators, as well as the usethereof, are described in our co-pending Patent Application Ser. No.892,362, filed Mar. 31, 1978, the arylsemicarbazides in question beingcompounds of the general formula:

    Ar-NH-NH-CO-NH.sub.2                                       (I)

wherein Ar is an aryl radical optionally substituted by alkyl, alkoxy orhalogen.

The aryl radical in the compounds (I) is preferably a phenyl or naphthylradical. The alkyl and alkoxy radicals can contain up to 4 carbon atoms,methyl or ethyl radicals being preferred, and the halogen atom ispreferably a fluorine, chlorine or bromine atom.

Furthermore, it is advantageous to impregnate the test papers with acomplex former in order to form a complex with the metal ions which areusually present in papers. The salts of ethylenediamine-tetraaceticacid, especially the potassium salt, have proved to be especiallysuitable for this purpose, particularly since they can, at the sametime, be used as buffers.

Because of the relatively large amounts of water-soluble substancespresent in the test papers, they can have a tendency to bleed so that itis advisable to add to the formulation thickening agents, such as methylcellulose, gelatine or polyvinyl pyrrolidone, which can also act asprotective colloids.

As wetting agents, there can be used, for example, long-chained organicsulphates or sulphonates.

For the production of the test papers according to the presentinvention, absorbent carriers, for example filter paper, cellulose orsynthetic resin fleece, are impregnated with solutions which contain thecomponents, preferably in mixtures of water and lower alcohols oracetone, whereafter the papers are dried.

However, it is, for example, also possible first to impregnate thecomplex former from water and then to impregnate the other componentsfrom organic solutions. In these papers, the most varied protectionbatches give the same results. In contradistinction thereto, batches ofpapers which have been impregnated with unpurified guaiac resinnaturally show considerable variations in their sensitivity.

In contradistinction to conventional test strips, in the case of thetest according to the present invention, not all of the reactioncomponents are impregnated on to the carrier. Thus, for reasons ofstability, it has been shown that the addition of hydrogen peroxide isonly to take place when an immediate evaluation of the coloration isensured. For this purpose, the detection of occult blood in feces iscarried out as follows: at test strip, the production of which isdescribed in the Examples given hereinafter, is first coated with asample of the feces to be investigated. After drying the sample, asolution of hydrogen peroxide in alcohol is applied dropwise to the rearside of the carrier and, when blood is present in the sample, a bluecoloration appears.

The following Examples are given for the purpose of illustrating thepresent invention:

EXAMPLE 1 Preparation of guaiaconic acid A

200 g. of ground guaiac resin are stirred into 500 ml. acetone, theresin, apart from a small amount thereof, thereby going into solution.While stirring, 3 liters of toluene are now added thereto dropwise, theprecipitated material (about 60 g.) is filtered off with suction afterabout 30 minutes and this material is discarded. The filtrate isevaporated in a vacuum, about 160 g. of a dark brown, glassy residuebeing obtained. This is dissolved in 500 ml. of an n-heptane/ethylacetate mixture (2:5 v/v), with warming, and, after cooling, it isapplied to a column of silica gel (3.1 kg. silica gel, particle size0.063-0.2 mesh), the column having a diameter of 80 cm. and a height of1.65 m., the silica gel used having previously been freed from iron with2 N hydrochloric acid, washed neutral with water and dried in a vacuum.Separation takes place with 7.5 liters of an n-heptane/ethyl acetatemixture (2:5 v/v) under carbon dioxide as protective gas. 150 fractions,each of 50 ml., are collected. Fractions 115-145 give, afterevaporation, about 16.5 g. of a beige-colored glassy material from whichabout 12 g. guaiaconic acid A are obtained by recrystallization from 150ml. xylene. It is preferable to investigate the individual fractions bythin layer chromatography since, under certain circumstances,displacements of the elution of the desired material can occur.

Thin layer chromatography

Silica gel finished plates 60 F-254 (Merck). Elution agent:toluene/dioxan/glacial acetic acid (90:25:10 v/v/v); R_(F) value 0.45.

Spray solution

With 0.05% peroxidase and 0.5% hydrogen peroxide, the guaiaconic acid Abecomes blue.

With 6% formalin and 26% sulphuric acid in water and heating for 5minutes, guaiaconic acid A becomes brown-violet and the impuritiesbecome red to violet.

Determination of the specific extinction

4.0 g. Guaiaconic acid A are dissolved in 400 ml. 50% aqueous alcohol.1.0 ml. of this solution, 0.5 ml. of a 3% solution of polyvinylpyrrolidone K 90 in distilled water, 1.0 ml. of a 0.1 molar aqueoussolution of hydrogen peroxide and 0.1 ml. of a 1% solution of horseradish peroxidase (1-2×10³ U/g.) in distilled water are made up to 10ml. with distilled water. The solution is well mixed up and, after 5 to10 minutes, measured in a 1 cm. cuvette at 600 nm.

Specific extinction=measured extinction×1000

Infra-red bands (KBr): 1600 cm⁻¹ (m); 1505 cm⁻¹ (v.s.); 1260 cm⁻¹ (s;b);1115 cm⁻¹ (m); 1025 cm⁻¹ (m); and 1200 cm⁻¹ (s;b).

EXAMPLE 2 Test paper for the detection of occult blood in feces

Filter paper (Schleicher & Schull 597 NF-Ind) is impregnated with thefollowing solution and dried at 50° C.:

    ______________________________________                                        guaiaconic acid A; E.sup.1%.sub.1 cm.  = 270 at 600 nm                                                 120     mg.                                          1-phenylsemicarbazide    65      mg.                                          polyvinyl pyrrolidone K 25                                                                             300     mg.                                          potassium EDTA buffer, pH 5.5*                                                                         10      ml.                                          acetone                  50      ml.                                          water                    ad 100  ml.                                          ______________________________________                                         *10 g. ethylenediaminetetraacetic acid +4.75 g. potassium hydroxide in 10     ml. water                                                                

To 30 samples of feces from different subjects there were admixedincreasing amounts of blood and the samples were homogenized. Understandardized conditions, the same amounts of samples were applied to thetest papers and, after drying, 2 drops of a mixture of 11 ml. perhydroland 100 ml. ethanol were applied to the rear side of the test papers.The following Table shows the percentage of negative and positivereactions, in comparison with a commercially available test paper:

                  TABLE                                                           ______________________________________                                                 test according to the                                                                        commercially available                                amount of                                                                              invention      product                                               blood    negative positive  negative positive                                 added    reaction reaction  reaction reaction                                 ______________________________________                                        1%       25       75        80       20                                       2%       10       90        72       18                                       3%       8        92        57       43                                       4%       0        100       50       50                                       5%       0        100       40       60                                       6%       0        100       40       60                                       ______________________________________                                    

The above Table shows that the practical limit of detection (i.e. thatconcentration of the material to be detected with which there isobtained a positive reaction in 90 cases out of 100) is, in the case ofthe test papers according to the present invention, present in the caseof an addition of 2% of blood. A practical limit of detection for thecommercially available product cannot be given, which is confirmed bythe manufacturer in his information leaflet. With aqueous bloodsolutions, both test papers react the same positively up to a dilutionof 1:5000.

EXAMPLE 3 Test paper for the detection of occult blood in feces

60 mg Guaiaconic acid A, E₁ cm.^(1%) =300 at 600 nm., are dissolved inethanol. Filter paper (Whatman No. 1) is impregnated with this solution.After drying at 50° C., a test paper is obtained which has practicallythe same properties as those described in Example 2.

EXAMPLE 4

140 mg. guaiaconic acid A, E₁ cm.^(1%) =240 at 600 nm, and 70 mg. of oneof the 1-arylsemicarbazides given in the following Table are dissolvedin 100 ml. methanol. Filter paper (Whatman No. 1) is impregnated withthis solution, the filter paper having been previously impregnated witha 0.4 molar buffer of ethylenediamine-tetraacetic acid and aqueoussodium hydroxide solution of pH 5.5 After drying at 50° C., test papersare obtained which have practically the same properties as thosedescribed in Example 2.

The following arylsemicarbazides were used:

1-phenylsemicarbazide;

1-(o-tolyl)-semicarbazide;

1-(m-tolyl)-semicarbazide;

1-(o-methoxyphenyl)-semicarbazide;

1-(p-methoxyphenyl)-semicarbazide;

1-(m-chlorophenyl)-semicarbazide;

1-(α-naphthyl)-semicarbazide.

It will be understood that the specification and examples areillustrative, but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. Diagnostic agent for the detection of occultblood in feces, comprising hydrogen peroxide and, as a chromogen,guaiaconic acid A, having a specific extinction E₁ cm^(1%) at 600 nm ofat least 200, determined by the reaction with peroxidase and hydrogenperoxide, having infra-red bands at1600 cm⁻¹ (m); 1505 cm⁻¹ (v.s.); 1260cm⁻¹ (s;b); 1115 cm⁻¹ (m); 1025 cm⁻¹ (m); and 1200 cm⁻¹ (s;b)and havingan R_(F) value of 0.45 (toluene/dioxan/glacial acetic acid; 90:25:10v/v/v).
 2. Diagnostic agent as claimed in claim 1 also comprising atleast one adjuvant selected from buffers, wetting agents, thickeningagents, protective colloids, complex formers and stabilizers. 3.Diagnostic agent as claimed in claim 1 comprising additionally abibulous carrier for said hydrogen peroxide and guaiaconic acid A.
 4. Ina method for the detection of occult blood in feces, using the reactionof hydrogen peroxide with a chromogen catalyzed by peroxidate-activecomponents of blood, by evaluation of the coloration, the improvementcomprising using, as the chromogen, guaiaconic acid A, having a specificextinction E₁ cm^(1%) at 600 nm of at least 200, determined by thereaction with peroxidase and hydrogen peroxide, having infra-red bandsat1600 cm⁻¹ (m); 1505 cm⁻¹ (v.s.); 1260 cm⁻¹ (s;b); 1115 cm⁻¹ (m); 1025cm⁻¹ (m); and 1200 cm⁻¹ (s;b)and having an R_(F) value of 0.45(toluene/dioxan/glacial acetic acid; 90:25:10 v/v/v).
 5. In a method forthe detection of occult blood in feces, wherein a sample of feces isfirst applied to an absorbent paper which contains a chromogen and atleast one adjuvant selected from buffers, wetting agents, thickeningagents, protective colloids, complex formers and stabilizers, and afterdrying an alcoholic solution of hydrogen peroxide is applied dropwise tothe rear side of the carrier and subsequently the coloration due to thereaction of the hydrogen peroxide with the chromogen catalyzed by theperoxidate-active blood components is evaluated, the improvementcomprising using, as the chromogen, guaiaconic acid A, having a specificextinction E₁ ^(1%) cm at 600 nm of at least 200, determined by thereaction with peroxidase and hydrogen peroxide, having infra-red bandsat1600 cm⁻¹ (m); 1505cm⁻¹ (v.s.); 1260 cm⁻¹ (s;b); 1115 cm⁻¹ (m); 1025cm⁻¹ (m); and 1200 cm⁻¹ (s;b)and having an R_(F) value of 0.45(toluene/dioxan/glacial acetic acid; 90:25:10 v/v/v.